The overall goal of this proposal is to exploit our understanding of the Ral signaling cascade to reveal how it contributes to cancer through its functions in both tumor cells and adjacent stromal fibroblasts. RalA and RalB are members of the Ras superfamily that become activated by a distinct set of guanine nucleotide exchange factors (Ral-GEFs), like RalGDS, in response to a variety of extracellular signals. Once activated, Ral proteins influence a unique set of downstream signaling molecules that regulate multiple cellular processes including delivery of membrane proteins like E-cadherin to the basolateral plasma membrane of epithelial cells, exocytosis, apoptosis, cell migration and cell proliferation. A key property of the Ral signaling cascade is that it is stimulated by Ras proteins. In many studies, including those involving RalGDS knockout mice, suppression of the Ral signaling cascade inhibits Ras-induced oncogenic transformation, however the mechanisms involved are poorly understood. Thus, there is intense interest in how the Ral signaling cascade is regulated and how its downstream effectors function in cancer. We recently discovered a new function for RalGDS as a scaffold that regulates Akt activation in mammary epithelial cells by growth factors. In Specific Aim 1, we will use a 3-D culture model of mammary gland development to test the hypothesis that RalGDS contributes to early steps in breast cancer development through it ability to regulate specific Akt family members. Using a bioengineered model of human skin, we found that RalA plays a cell-type dependent role in Ras-mediated squamous cell carcinoma. In keratinocytes of the epithelium, RalA inhibits tumorigenesis, since suppression of RalA expression enhances tumor progression. Specific Aim 2 will focus on how RalA displays this effect through its regulation of E-cadherin. In fibroblasts of the dermis RalA supports cancer progression, since RalA knockdown in these cells suppresses progression of adjacent tumorigenic keratinoctyes. These findings suggest that the tumor-resistant phenotype of RalGDS knockout mice is due, at least in part, to inhibition of fibroblast RalA. They also raise the exciting possibility of targeting the Ral signaling cascade in genetically stable dermal fibroblasts, which have a low probability of developing drug resistance, as a novel mode of anti-cancer therapy. Specific Aim 3 will reveal how knockdown of RalA in stromal fibroblasts blocks tumor progression of adjacent keratinocytes in these engineered tissues. It will also test whether knockdown of other components of the Ral signaling cascade in fibroblasts also blocks tumor progression, and whether these effects can be recapitulated in mice.